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1: Dis Aquat Organ. 2001 Oct 29;47(1):13-23. Related Articles, Links

Development and application of monoclonal antibodies for the detection of white spot syndrome virus of penaeid shrimp.

Poulos BT, Pantoja CR, Bradley-Dunlop D, Aguilar J, Lightner DV.

University of Arizona, Department of Veterinary Science and Microbiology, Tucson 85721, USA. bpoulos@u.arizona.edu

Monoclonal antibodies (MAbs) were produced against white spot syndrome virus (WSSV) of penaeid shrimp. The virus isolate used for immunization was obtained from China in 1994 and was passaged in Penaeus vannamei. The 4 hybridomas selected for characterization all produced MAbs that reacted with the 28 kD structural protein by Western blot analysis. The MAbs tested in dot-immunoblot assays were capable of detecting the virus in hemolymph samples collected from moribund shrimp during an experimentally induced WSSV infection. Two of the MAbs were chosen for development of serological detection methods for WSSV. The 2 MAbs detected WSSV infections in fresh tissue impression smears using a fluorescent antibody for final detection. A rapid immunohistochemical method using the MAbs on Davidson's fixed tissue sections identified WSSV-infected cells and tissues in a pattern similar to that seen with digoxigenin-labeled WSSV-specific gene probes. A whole mount assay of pieces of fixed tissue without paraffin embedding and sectioning was also successfully used for detecting the virus. None of the MAbs reacted with hemolymph from specific pathogen-free shrimp or from shrimp infected with infectious hypodermal and hematopoietic necrosis virus, yellow head virus or Taura syndrome virus. In Western blot analysis, the 2 MAbs did not detect any serological differences among WSSV isolates from China, Thailand, India, Texas, South Carolina or Panama. Additionally, the MAbs did not detect a serological difference between WSSV isolated from penaeid shrimp and WSSV isolated from freshwater crayfish.

PMID: 11797911 [PubMed - indexed for MEDLINE]
 
1: Dis Aquat Organ. 2002 Aug 15;51(1):67-75. Related Articles, Links

Monoclonal antibodies developed for sensitive detection and comparison of white spot syndrome virus isolates in India.

Anil TM, Shankar KM, Mohan CV.

Department of Aquaculture, University of Agricultural Sciences, College of Fisheries, Mangalore, India.

Since its first report in 1994, white spot syndrome virus (WSSV) has become widespread in India. We have developed a simple, rapid and sensitive monoclonal antibody (MAb)-based immunodot test for detection of WSSV. Four MAbs of IgG isotype were produced against an Indian isolate of WSSV: 1 MAb recognised a 28 kDa viral protein while the other 3 recognised both 28 and 18 kDa proteins. The 4 MAbs recognised 4 different Indian WSSV isolates collected at different times from the east and west coasts of India, indicating antigenic uniformity of the isolates. The limit of detection of the immunodot test was 500 pg of the viral protein, which compared well with 1 step PCR and could be used to detect WSSV in shrimp Penaeus monodon with and without gross signs of white spots in the cuticle. Furthermore, the test was rapid (3 h for completion) and is suitable for further development as a simple field kit.

PMID: 12240972 [PubMed - indexed for MEDLINE]
 
1: Dis Aquat Organ. 2002 Apr 24;49(1):11-8. Related Articles, Links

Detection of white spot syndrome virus (WSSV) of shrimp by means of monoclonal antibodies (MAbs) specific to an envelope protein (28 kDa).

Liu W, Wang YT, Tian DS, Yin ZC, Kwang J.

Institute of Molecular Agrobiology, The National University of Singapore, Singapore.

The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.

PMID: 12093036 [PubMed - indexed for MEDLINE]
 
1: Dis Aquat Organ. 2000 Aug 10;42(1):27-34. Related Articles, Links

Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon.

Sithigorngul P, Chauychuwong P, Sithigorngul W, Longyant S, Chaivisuthangkura P, Menasveta P.

Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand. paisarn@psm.swu.ac.th

A monoclonal antibody specific to yellow head virus (YHV) was produced from a mouse immunized with gill extracts prepared from laboratory-reared Penaeus monodon dually infected with YHV and white spot syndrome virus (WSSV). One clone designated V3-2B specifically bound to native and SDS-treated viral specific antigens. Immunocytochemical studies of infected gills revealed viral specific immunoreactivities in the cytoplasm of gill tissue and in haemocytes. No antibody binding was observed in gills from non-infected shrimp. In addition, immunocytochemical examination of tissues from shrimp experimentally infected with YHV gave a positive reaction, while tissues from uninfected control shrimp or shrimp experimentally infected with WSSV did not. Western blot analysis indicated that the antibody reacted with a protein of approximately 135 kD that was present only in shrimp infected with YHV. In dot-blot indirect immunoperoxidase assays, the antibody was able to detect viral associated antigen in diluted haemolymph up to 1:50 dilution and in an ammonium sulfate precipitate of haemolymph up to 1:1000 dilution. The results suggested that this antibody might be useful for development of effective diagnostic techniques for both heavy and mild YHV infections in shrimp.

PMID: 10986642 [PubMed - indexed for MEDLINE]